Influenza is responsible for much morbidity and mortality in the world and is considered by many as belonging to the most significant viral threats to humans. Annual Influenza epidemics swipe the globe and occasional new virulent strains cause pandemics of great destructive power. At present the primary means of controlling Influenza virus epidemics is vaccination. However, mutant Influenza viruses are rapidly generated which escape the effects of vaccination. In the light of the fact that it takes approximately 6 months to generate a new Influenza vaccine, alternative therapeutic means, i.e., antiviral medication, are required especially as the first line of defense against a rapidly spreading pandemic.
An excellent starting point for the development of antiviral medication is structural data of essential viral proteins. Thus, the crystal structure determination of the Influenza virus surface antigen neuraminidase (von Itzstein et al., 1993, Nature 363:418-423) led directly to the development of neuraminidase inhibitors with anti-viral activity preventing the release of virus from the cells, however, not the virus production. These and their derivatives have subsequently developed into the anti-Influenza drugs, zanamivir (Glaxo) and oseltamivir (Roche), which are currently being stockpiled by many countries as a first line of defense against an eventual pandemic. However, these medicaments provide only a reduction in the duration of the clinical disease. Alternatively, other anti-Influenza compounds such as amantadine and rimantadine target an ion channel protein, i.e., the M2 protein, in the viral membrane interfering with the uncoating of the virus inside the cell. However, they have not been extensively used due to their side effects and the rapid development of resistant virus mutants (Magden et al., 2005, Appl. Microbiol. Biotechnol. 66:612-621). In addition, more unspecific viral drugs, such as ribavirin, have been shown to work for treatment of Influenza infections (Eriksson et al., 1977, Antimicrob. Agents Chemother. 11:946-951). However, ribavirin is only approved in a few countries, probably due to severe side effects (Furuta et al., 2005, Antimicrob. Agents Chemother. 49:981-986). Clearly, new antiviral compounds are needed, preferably directed against different targets.
Influenza virus A, B, C and Isavirus as well as Thogotovirus belong to the family of Orthomyxoviridae which, as well as the family of the Bunyaviridae, including the Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, and Tospovirus, are negative stranded RNA viruses. Their genome is segmented and comes in ribonucleoprotein particles that include the RNA dependent RNA polymerase which carries out (i) the initial copying of the single-stranded virion RNA (vRNA) into viral mRNAs and (ii) the vRNA replication. For the generation of viral mRNA the polymerase makes use of the so called “cap-snatching” mechanism (Plotch et al., 1981, Cell 23:847-858; Kukkonen et al., 2005, Arch. Virol. 150:533-556; Leahy et al., 1997, J. Virol. 71:8347-8351; Noah and Krug, 2005, Adv. Virus Res. 65:121-145). The polymerase is composed of three subunits: PB1 (polymerase basic protein), PB2, and PA. For the cap-snatching mechanism, the viral polymerase binds via its PB2 subunit to the 5′ RNA cap of cellular mRNA molecules which are cleaved at nucleotide 10 to 13 by the endonucleolytic activity of the polymerase. The capped RNA fragments serve as primers for the synthesis of viral mRNAs by the nucleotidyl-transferase center in the PB1 subunit (Li et al., 2001, EMBO J. 20:2078-2086). Finally, the viral mRNAs are 3′-end poly-adenylated by stuttering of the polymerase at an oligo-U motif at the 5′-end of the template. Recent studies have precisely defined the structural domain of PB2 responsible for cap-binding (Fechter et al., 2003, J. Biol. Chem. 278:20381-20388; Guilligay et al., 2008 Nat. Struct. Mol. Biol. 15:500-506). The endonucleolytic activity of the polymerase has hitherto been thought to reside in the PB1 subunit (Li et al, supra).
The polymerase complex seems to be an appropriate antiviral drug target since it is essential for synthesis of viral mRNA and viral replication and contains several functional active sites likely to be significantly different from those found in host cell proteins (Magden et al., supra). Thus, for example, there have been attempts to interfere with the assembly of polymerase subunits by a 25-amino-acid peptide resembling the PA-binding domain within PB1 (Ghanem et al., 2007, J. Virol. 81:7801-7804). Moreover, there have been attempts to interfere with viral transcription by nucleoside analogs, such as 2′-deoxy-2′-fluoroguanosine (Tisdale et al., 1995, Antimicrob. Agents Chemother. 39:2454-2458) and it has been shown that T-705, a substituted pyrazine compound may function as a specific inhibitor of Influenza virus RNA polymerase (Furuta et al., supra). Furthermore, the endonuclease activity of the polymerase has been targeted and a series of 4-substituted 2,4-dioxobutanoic acid compounds has been identified as selective inhibitors of this activity in Influenza viruses (Tomassini et al., 1994, Antimicrob. Agents Chemother. 38:2827-2837). In addition, flutimide, a substituted 2,6-diketopiperazine, identified in extracts of Delitschia confertaspora, a fungal species, has been shown to inhibit the endonuclease of Influenza virus (Tomassini et al., 1996, Antimicrob. Agents Chemother. 40:1189-1193). However, the inhibitory action of compounds on the endonucleolytic activity of the viral polymerase was hitherto only studied in the context of the entire trimeric complex of the polymerase.
The PA subunit of the polymerase is functionally the least well-characterised, although it has been implicated in both cap-binding and endonuclease activity, vRNA replication, and a controversial protease activity. PA (716 residues in influenza A) is separable by trypsination at residue 213. The recently determined crystal structure of the C-terminal two-thirds of PA bound to a PB1 N-terminal peptide provided the first structural insight into both a large part of the PA subunit, whose function, however, still remains unclear, and the exact nature of one of the critical inter-subunit interactions (He et al., 2008, Nature 454:1123-1126; Obayashi et al., 2008, Nature 454:1127-1131). Systematic mutation of conserved residues in the PA amino-terminal domain have identified residues important for protein stability, promoter binding, cap-binding and endonuclease activity of the polymerase complex (Hara et al., 2006, J. Virol. 80:7789-7798). The enzymology of the endonuclease within the context of intact viral ribonucleoprotein particles (RNPs) has been extensively studied.
It has been found recently that, contrary to the general opinion in the field, the endonucleolytic activity resides exclusively within the PA subunit of the RNA-dependent RNA polymerase of the Influenza A H3N2 virus (Dias et al., 2009).
The present inventors have now achieved to structurally characterize the PA domain of the Influenza A 2009 pandemic H1N1 virus by X-ray crystallography and identified the endonucleolytic active center within said domain. The present inventors surprisingly found that polypeptide fragments of the PA subunit of said virus readily crystallized and that, thus, said polypeptide fragments are very suitable to study the endonucleolytic activity of the RNA-dependent RNA polymerase of the Influenza A 2009 pandemic H1N1 virus in the context of said polypeptide fragments in order to simplify the development of new anti-viral compounds targeting the endonuclease activity of said viral polymerase as well as to optimize previously identified compounds.
The achievement of the present inventors to recombinantly produce PA polypeptide fragments possessing the endonucleolytic activity of the RNA-dependent RNA polymerase of the Influenza A 2009 pandemic H1N1 virus allows for performing in vitro high-throughput screening for inhibitors of a functional site on said viral polymerase using easily obtainable material from a straightforward expression system. Furthermore, the structural data of the endonucleolytic PA H1N1 polypeptide fragment as well as of the enzymatically active center therein allows for directed design of inhibitors and in silico screening for potentially therapeutic compounds.
The present inventors further managed, for the first time, the co-crystallization of a PA polypeptide fragment and of a variant thereof of the PA subunit of Influenza A 2009 pandemic H1N1 virus with a bound inhibitor and found that, thus, the development of new anti-viral compounds targeting the endonuclease activity of the RNA-dependent RNA polymerase of the Influenza A 2009 pandemic H1N1 virus can be improved. Particularly, the co-crystallization data show, for the first time, in detail which amino acids comprised in the active site of a PA polypeptide fragment of the PA subunit of Influenza A 2009 pandemic H1N1 virus are especially involved in compound binding. This new knowledge allows the optimized design of modifications to existing inhibitors in order to improve their potency or the design and optimization of novel inhibitors that effectively block endonuclease activity.
It is an object of the present invention to provide (i) high resolution structural data of the endonucleolytic amino-terminal domain of the viral polymerase H1N1 PA subunit by X-ray crystallography, (ii) high resolution structural data of the endonucleolytic amino-terminal domain of the viral polymerase H1N1 PA subunit co-crystallized with a known inhibitor by X-ray crystallography, (iii) computational as well as in vitro methods, preferably in a high-throughput setting, for identifying compounds that can modulate, preferably inhibit, the endonuclease activity of the viral polymerase of the Influenza A 2009 pandemic H1N1 virus, preferably by blocking the endonucleolytic active site within the H1N1 PA subunit, and (iv) pharmacological compositions comprising such compounds for the treatment of infectious diseases caused by viruses using the cap snatching mechanism for synthesis of viral mRNA.